The vir locus of Bordetella pertussis is a central regulatory gene which controls the expression of pertussis toxin, FHA, and all other protein virulence determinants in response to environmental stimuli. Our goal is to understand the molecular mechansims by which this regulation occurs. To aid in our genetic analyses we have recently constructed strains of B. pertussis containing gene fusions such that FHA expression is measured in terms of b-galactosidase production, and pertussis toxin in terms of alkaline phosphatase production. These strains are proving useful in the quantitation of the effects of mutations in the vir locus. We have identified several genetic situations in which there is differential expression of pertussis toxin and FHA, suggesting different levels of genetic control of these two virulence determinants. Our biochemical analysis of the proteins encoded by vir: BvgS and BvgA, have advanced to the point where we have purified BvgA, as well as a MBP:BvgS fusion protein. This has allowed us to begin in vitro studies of the hypothesized protein kinase/phosphoacceptor activities of this protein pair. We have also begun an analysis of the B. pertussis chromosome with the goal of constructing a physical map. This analysis is almost complete and will allow us to determine if the genes for virulence determinants of B. pertussis are linked in an evolutionarily relevant fashion.